The ideal method for imaging living cells is one that allows for long-term,label-free observation in real-time with super-resolution capabilities.Such a method would overcome the drawbacks of phototoxicity and photobl...The ideal method for imaging living cells is one that allows for long-term,label-free observation in real-time with super-resolution capabilities.Such a method would overcome the drawbacks of phototoxicity and photobleaching associated with fluorescence labeling microscopy.Fourier ptychography is a promising label-free imaging technique that surpasses the diffraction limit of conventional microscopy while avoiding issues related to fluorescent labeling.However,previous Fourier ptychography microscopy (FPM) systems lacked high spatiotemporal resolution,preventing real-time observation of subcellular organelle structures in living cells.To address this limitation,we have developed a high-speed super-resolution microscope based hemispherical digital illumination (HDI) device.This system utilizes 61 light-emitting diodes (LEDs) to provide high-angle illumination with a numerical aperture (NA) of 0.98.By employing a 40×/0.6 NA objective lens,we have achieved lateral resolutions of around 150 nm,enabling us to capture images at a speed of over 1 Hz with the field of view measuring 118×118 μm2.HDI-FPM allows for the monitoring of various cellular processes,such as vesicular transportation,mitochondrial fusion and division,as well as cell-cell fusion over a duration of 4 h.The development of HDI-FPM represents a significant advancement in label-free imaging of living cells.It offers a comprehensive understanding of the mechanisms underlying cellular activities.展开更多
基金supported by the Scientific Instrument Developing Project of the Chinese Academy of Sciences(No.ZDKYYQ20220005)the National Natural Science Foundation of China(Nos.22150003,and 22327808)Chinese patent applications have been submitted for relevant technologies(No.202011538556.8,No.202110126844.0&No.ZL 202120587363.5).
文摘The ideal method for imaging living cells is one that allows for long-term,label-free observation in real-time with super-resolution capabilities.Such a method would overcome the drawbacks of phototoxicity and photobleaching associated with fluorescence labeling microscopy.Fourier ptychography is a promising label-free imaging technique that surpasses the diffraction limit of conventional microscopy while avoiding issues related to fluorescent labeling.However,previous Fourier ptychography microscopy (FPM) systems lacked high spatiotemporal resolution,preventing real-time observation of subcellular organelle structures in living cells.To address this limitation,we have developed a high-speed super-resolution microscope based hemispherical digital illumination (HDI) device.This system utilizes 61 light-emitting diodes (LEDs) to provide high-angle illumination with a numerical aperture (NA) of 0.98.By employing a 40×/0.6 NA objective lens,we have achieved lateral resolutions of around 150 nm,enabling us to capture images at a speed of over 1 Hz with the field of view measuring 118×118 μm2.HDI-FPM allows for the monitoring of various cellular processes,such as vesicular transportation,mitochondrial fusion and division,as well as cell-cell fusion over a duration of 4 h.The development of HDI-FPM represents a significant advancement in label-free imaging of living cells.It offers a comprehensive understanding of the mechanisms underlying cellular activities.
